To understand the mechanism of smooth muscle contraction and how actin and myosin interact in nonmuscle cells, we have been using various assays of myosin function. One of these assays involves the visualization in the fluorescent microscope of the movement of fluorescently-labeled actin filaments over a surface coated with myosin molecules. This movement is an active process which requires the presence of myosin and MgATP. The movement of actin filaments by smooth muscle and nonmuscle myosins is almost completely dependent upon phosphorylation of the myosin by myosin light chain kinase. Turkey gizzard smooth muscle myosin translocates actin filaments at about 5 times the rate of translocation obtained with human platelet myosin. Myosin from bovine brain appears to move actin filaments even more slowly than does platelet myosin. Heavy meromyosin, the soluble two-headed proteolytic fragment of myosin, can be prepared from platelet myosin. This heavy meromyosin can also translocate actin filaments in the motility assay at a rate similar to that obtained with the intact platelet myosin. Myosin can be bound to the glass surface as either filaments (consisting of a packed array of myosin molecules) or as monomers. Both forms translocate actin filaments at the same rate indicating that with smooth muscle and vertebrate nonmuscle myosin, filaments are not absolutely required for activity. This also raises the possibility that myosin filaments per se may not be necessary for motile functions in cells and may explain some of the difficulties that have been experienced in demonstrating the presence of myosin thick filaments in vertebrate nonmuscle cells. In order to better understand the role filaments play in the interaction of smooth muscle myosin with actin, we have cross-linked the myosin molecules within a filament with EDC. These cross-linked myosin filaments do not depolymerize under conditions that normally promote depolymerization of myosin and, thus, represent a mechanism for studying actin-myosin filament interactions under conditions where the filaments are unstable in vitro.